CN102277373A - Schistosoma japonica vaccine expression vector for attenuated salmonella secretion expression and use thereof - Google Patents
Schistosoma japonica vaccine expression vector for attenuated salmonella secretion expression and use thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and particularly relates to a schistosoma japonica vaccine salmonella secretion expression vector, which is a recombinant salmonella expression vector that is controlled by a bacterial promoter, guided by effector protein secreted by salmonella and secretion signal of the effector protein and used for secretion expression of schistosoma japonica antigen. The schistosoma japonica vaccine salmonella secretion expression vector can induce secretion expression of a schistosoma japonica vaccine in a hypoxic microenvironment in antigen presenting cells, can exist in vivo stably and continuously and is insusceptible to being lost. The schistosoma japonica vaccine salmonella secretion expression vector, by using an attenuated strain as a carrier, can perform the antigen carrying of the recombinant vaccine representing bacteria by means of oral taking. The schistosoma japonica vaccine salmonella secretion expression vector can be used for preparing schistosoma japonica vaccine for oral taking for preventing and treating schistosomiasis.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Japanese blood fluke vaccine expression vector and application thereof of attenuation salmonella secreting, expressing.
Background technology
Schistosomicide is the infecting both domestic animals and human parasitosis of a kind of serious harm mankind and animal health, and about 600,000,000 people in the whole world live in the schistosomicide epidemic-stricken area, and 200,000,000 people are infected, and clinical symptom appears in 1.2 hundred million people, and year death toll is nearly hundreds thousand of.After working out the specifics praziquantel, by colony's chemotherapy, to popular existing alleviation of schistosomicide, but the propagation of schistosomicide is still continuing, and number of patients is still increasing.And the resistance of chemicals such as praziquantel has also influenced control (McManus DP, Clin Microbiol Rev, 2008,21 (1): 225-242) of schistosomicide to a great extent.Therefore, for the propagation of better prevention and cure of schistosomiasis and popular, need a kind of efficient, safe, inexpensive blood fluke vaccine of exploitation in a hurry.
Blood fluke vaccine research has successively been experienced the stages such as killed vaccine, deactivation or attenuated vaccine, recombiant vaccine and nucleic acid vaccine.Though inactivated vaccine has shown higher protection, its risk height; It is not high that killed vaccine, recombiant vaccine and nucleic acid vaccine but are faced with protection, involves great expense inoculation inconvenience or the like problem.Oral antigen submission system has inoculation simply because of it, and advantages of cheap price is widely used in multiple infectious diseases vaccine development.The Salmonellas of active attenuation is a kind of ideal oral vaccine carrier, but its by oral route, the exogenous antigen molecule is transported in the antigen presenting cell (APC), and pass through histocompatibility complex's submission and give the T cell, excitating organism produces cell, body fluid and the mucosal immune response at antigen molecule.
Khan CM attempts utilizing Salmonellas attenuated strain SL3261 to express Schistosoma mansoni 28kd GST antigen first, behind the oral immunity mouse, in serum, detected at the antigenic antibody of GST (Khan CM, Proc Natl Acad Sci. 1994,91 (23): 11261-11265); Pacheco LG etc. utilizes attenuation salmonella SL3261 to express Schistosoma mansoni 14 kd lipid acid albumen as a result, and (Acta Trop. 2005,95 (2): 132-142) to have obtained 34.9% worm reduction rate behind the oral mouse.After but in a single day Salmonellas enters antigen presenting cell, just be limited in a kind of film parcel vesica (Salmonella containing vacuole, SCV) among (Zhang XL etc., Cell Mol Immunol. 2008,5 (2): 91-97).The vesica structure of this film parcel has limited antigenic effective submission to a great extent.Therefore, in order to break this restriction, developing a kind of is the blood fluke vaccine of carrier with oral attenuation salmonella efficiently, need face the secretion problem of antigen molecule.
Therefore, set up an attenuation salmonella secreting, expressing Schistosoma japonicum oral vaccine system efficiently, must make up a Salmonellas secretion expression carrier efficiently, to realize the secreting, expressing of antigen molecule in Salmonellas, this is a problem to be solved in the present invention.
Summary of the invention
The problem that the present invention need solve is to set up an attenuation salmonella secreting, expressing Schistosoma japonicum oral vaccine system efficiently, makes up a kind of secretion expression carrier of Salmonellas efficiently, to realize the secreting, expressing of antigen molecule in Salmonellas.
Main purpose of the present invention is to make up Salmonellas secretion expression carrier efficiently; realize the stable in vivo secreting, expressing that continues of antigen molecule, for set up express stable, protectiveness efficient is high is that the Schistosoma japonicum oral vaccine of transport agent lays the foundation with the attenuation salmonella.
In order to achieve the above object, the present invention utilizes III type excretory system and hemolysin excretory system that the schistosome antigen molecule is secreted, III type secretion signal and hemolysin secretion signal and antigen molecule merged realize comprising the antigenic secretion of Schistosoma japonicum, described III type secretion signal can be the secretory signal sequence of protokaryon bacterium III type secretion effect protein such as Salmonellas outer membrane protein E N end 1-104 aminoacid sequence; Described hemolysin secretion signal is 34 amino acid of hlyA albumen n end and 61 aminoacid sequences of C end; Described antigen molecule is vaccine antigens such as the two valency antigens of sj23LHD-GST.
Further, the invention provides a kind of schistosomicide oral vaccine submission system, it is characterized in that with the attenuation salmonella being the oral vaccine delivery vehicles, with the mode submission Japan schistosome antigen molecule of secreting, expressing, described attenuated bacteria strain can be attenuation salmonella VNP20009.
Further, the invention provides a kind of stable be the Schistosoma japonicum secreting, expressing oral vaccine of carrier with the attenuation salmonella, plasmid is expressed stable in vivo, does not lose.
Compare with existing blood fluke vaccine, characteristic of the present invention and innovation part are:
(1) invented a kind of Salmonellas secreting, expressing signals direct Japanese blood fluke vaccine antigen expression vector that utilizes, the two antigenic secreting, expressings of valency of Schistosoma japonicum have been realized, and confirm to utilize this system at mouse model, can realize that the liver worm reduction rate reaches 41.69%, the liver egg reduction rate is 57.71%.
(2) invented a kind of Japanese blood fluke vaccine of salmonella-mediating of oral form, and can obtain higher humoral immunization and cell immune response at antigen molecule at oral this vaccine of confirmations such as animal infection modal.
(3) invented a kind of Japanese blood fluke vaccine with low cost, the present invention owing to invented a kind ofly efficiently expresses, efficient excretory Japanese blood fluke vaccine antigen expression vector, add and utilize the convenient Salmonellas of cultivating, have the application prospect of large-scale promotion as antigen presentation factory and submission instrument.
In sum, the invention provides a kind of brand-new, efficiently, safety, cheap, easily, oral, can be used for the Japanese blood fluke vaccine secretion expression carrier and the application thereof of humans and animals.
Four, description of drawings
The structure of figure one, attenuation salmonella secretion expression carrier
(1A) the big hydrophilic area and the gst fusion protein of schistosome antigen sj23LHDGST(sj23 molecule) the structure synoptic diagram of secretion expression carrier.Two kinds of excretory systems have been made up.1, III type secreting, expressing system; 2, hemolysin secreting, expressing system.PnirB and phlyA are respectively nitrite reductase promotor and hemolysin gene A(hlyA) promotor; SopE1-104 is a Salmonellas III type excretory system secretion signal; HlyA is the hemolysin secretion signal, and hlyB, hlyC, hlyD are the required element of hemolysin excretory system.
(1B) situation of the recombinant salmonella secreting, expressing antigen molecule of Western bolting checking secreting, expressing to the nutrient solution supernatant.1, unloaded bacterium; 2, reorganization hemolysin secretion bacterium; 3, reorganization III type secretion bacterium is normally cultivated; 4, reorganization III type secretion bacterium anaerobism is cultivated.III type secretion bacterium is by the nirB promoter expression antigen of anaerobism regulation and control, and it is expressed needs weary oxygen environment to induce.
(1C) recombinant salmonella of secreting, expressing infects behind antigen presenting cell (mouse macrophage RAW264.7), analogue antigen EGFP submission is given the expression of scavenger cell.DAPI dyeing expression nucleus, 1, unloaded bacterium; 2, reorganization III type secretion bacterium; 3, reorganization hemolysin secretion bacterium.
The recombinant attenuated Salmonellas blood fluke vaccine of figure two, secreting, expressing is titre and time length in vivo
(2A) BALB/C mice oral 10
9Behind the recombinant vaccine strain of colony-forming unit (cfu) secreting, expressing and the wild bacterium in 3-20 days, the titre of reorganization bacterium in the peripheral immune organ spleen.1, wild bacterium (non-conversion Salmonellas) is represented total count; 2, reorganization hemolysin secretion bacterium; 3, reorganization III type secretion bacterium.The recombinant salmonella of secreting, expressing continues to exist in spleen, and the vaccine plasmid is not lost.
(2B) BALB/C mice oral 10
9Behind the recombinant salmonella of cfu secreting, expressing and the wild bacterium in 3-20 days, the titre of reorganization bacterium in the peripheral immune organ lymphoglandula.1, wild bacterium (non-conversion Salmonellas) is represented total count; 2, reorganization hemolysin secretion bacterium; 3, reorganization III type secretion bacterium.The recombinant salmonella of secreting, expressing continues to exist in lymphoglandula, and the vaccine plasmid is not lost.
The immune response that body produces behind the recombiant vaccine bacterium of figure three, BALB/C mice oral vaccination secreting, expressing at antigen molecule sj23LHDGST.
(3A) the oral unloaded bacterium of mouse, reorganization III type secretion bacterium and reorganization hemolysin are secreted bacterium three times, and at interval biweekly, a week is got serum after the last immunity, the antigenic IgG antibody titer of anti-sj23LHDGST in the serum analysis.1, unloaded bacterium; 2, reorganization III type secretion bacterium; 3, reorganization hemolysin secretion bacterium.
(3B) mouse oral immunity PBS, unloaded bacterium, reorganization hemolysin secretion bacterium and reorganization III type secretion bacterium (10
9Cfu) three times, at interval biweekly, a week is got spleen after the last immunity, and through behind the erythrocyte splitting, the preparation splenocyte suspension is used recombinant antigen sj23LHD, GST then respectively, and positive control ConA stimulated analysis splenocyte secretion of gamma-IFN amount 72 hours.1, PBS; 2, unloaded bacterium; 3, reorganization hemolysin secretion bacterium; 4, reorganization III type secretion bacterium.
The subcutaneous infection of reorganization blood fluke vaccine bacterium postabdomen Schistosoma japonicum protectiveness efficiency analysis after 42 days of figure four, mouse oral immunity secreting, expressing.
(4A) BALB/C mice oral immunity PBS, unloaded bacterium, reorganization hemolysin secretion bacterium and reorganization III type secretion bacterium (10
9Cfu) three times, at interval biweekly, in a week after the last immunity, subcutaneous abdomen infects 40 of schistosoma japonicum cercariaes, infects to calculate in back 42 days to become borer population in the mouse body.1, PBS; 2, unloaded bacterium; 3, reorganization hemolysin secretion bacterium; 4, reorganization III type secretion bacterium.
(4B) one-tenth borer population * 100% of worm reduction rate=(the average every mouse of one-tenth borer population-experimental group of the average every mouse of control group becomes borer population)/average every mouse of control group.1, unloaded bacterium; 2, reorganization hemolysin secretion bacterium; 3, reorganization III type secretion bacterium.
(4C) method of utilizing 5% potassium hydroxide (KOH) digestion liver to spend the night is calculated liver worm's ovum number.1, PBS; 2, unloaded bacterium; 3, reorganization hemolysin secretion bacterium; 4, reorganization III type secretion bacterium.
(4D) egg reduction rate=(the average every mouse liver worm's ovum number of the average every mouse liver worm's ovum number-experimental group of the control group)/average every mouse liver worm's ovum number of control group * 100%.1, unloaded bacterium; 2, reorganization hemolysin secretion bacterium; 3, reorganization III type secretion bacterium.
Five, embodiment:
Big hydrophilic area and gst fusion protein with the two valency antigen sj23LHDGST(sj23 molecules of Schistosoma japonicum) be embodiment as delivering antigen:
1, the structure of the vaccine carrier of secreting, expressing:
Antigen molecule sj23LHD-GST adopts Salmonellas III type excretory system and hemolysin excretory system to secrete respectively.
A) structure of III type secretion expression carrier: by 1-104 amino acid (sopE of III type secretion effector molecule sopE N end
1-104) realize its secretion with antigen molecule sj23LHD coupling; Adopt intestinal bacteria nitrate reductases (nirB) promotor that III type secretion antigen is expressed.The nirB promotor utilizes PCR method to obtain: primer sequence is: P1:5 '-cccctcgagggttaccggcccgatcg-3 '; P2:5 '-cccggatccaccgcctaccttaacgattc-3 '; With 50 ng bacillus coli gene group DNA as template.The fragment two ends that obtain have Xho I and BamH I restriction enzyme site, and the PCR program all is 94 ℃ of 40 s, 60 ℃ of 40 s, 72 ℃ of 1 min, 26 circulations.Salmonellas III type excretory system secretion signal is the amplification of sopE albumen 1-104 aminoacid sequence gene: primer sequence is: P1:5 '-cccggatccatgactaacataacactatc-3 '; P2:5 '-aaaggtacccggatctttactcgcat-3 '; Resulting sopE1-104 gene two ends have the restriction enzyme site of BamH I and Kpn I respectively, and the PCR program all is 94 ℃ of 40 s, 60 ℃ of 40 s, 72 ℃ of 1 min, 26 circulations.The acquisition of bivalent vaccine gene sj23LHDGST: adopt overlapping PCR method amplification to obtain the sj23LHDGST gene, primer sequence is: P1:5 '-aaaggtaccatgtacaaggataaaatcgatg-3 '; P2:5 '-taagttgcgttttaagaatgctagtataggggacat-3 '; P3:5 '-ttcttaaaacgcaacttaatgtcccctatactaggt-3 '; P4:5 '-cccaagcttttattttggaggatggtcgc-3 '; It is as follows to have method: earlier respectively with P1 and P2, P3 and P4 are that primer is a template with pcDNA3.1-sj23 and pcDNA3.1-GST, amplify sj23LHD fragment and GST fragment; Being primer with P1 and P4 then, is that template obtains sj23LHDGST bivalent vaccine gene with sj23LHD fragment and the GST fragment of expanding, and the sj23LHDGST gene two ends of being increased have Kpn I and Hind III restriction enzyme site respectively.The PCR program all is 94 ℃ of 40 s, 60 ℃ of 40 s, 72 ℃ of 1 min, 26 circulations.The connection of promotor-secretion signal-antigen gene: select low copy expression vector pQE30 for use, at first the nirB promotor is connected between the Xho I and BamH I of pQE30 carrier; Secondly with secretion signal sopE
1-104Insert between BamH I and the Kpn I site; Then bivalent vaccine antigen gene sj23LHDGST gene is inserted between Kpn I and the Hind III site.After transforming, identify, checking order, obtained III type secretion expression carrier nirB-sopE
1-104-sj23LHDGST.The enzyme program of cutting wherein is: 20 microlitre reaction systems, 37 ℃, 3 hours; Linker is: 10 microlitre reaction systems, room temperature, 5 hours.
B) the hemolysin excretory system is delivered the structure of vaccine antigen carrier: utilize hemolysin secretion expression carrier pMohly1 to realize antigenic hemolysin secreting, expressing, this carrier contains necessary all elements of hemolysin excretory system and secretion signal, and adopts the original promotor of effector molecule hlyA to express.The sj23LHDGST gene is connected in the Nsi I of pMohly1 single endonuclease digestion site, and with to read frame up and down consistent; The primer sequence of sj23LHDGST gene of being used to increase is: P1:5 '-ccaatgcatcgtacaaggataaaatcg-3 '; P2:5 '-ccaatgcatcttttggaggatggtcgc-3 '; Amplification PCR program is 94 ℃ of 40 s, 60 ℃ of 40 s, 26 circulations of 72 1 min.Then amplified production and carrier pMohly1 are carried out Nsi I enzyme and cut endonuclease reaction system 20 microlitres, 37 ℃, 3 hours; After enzyme cut product and reclaim, carrier carried out dephosphorization acid with alkaline phosphatase, and reaction system is 20 microlitres, and 37 ℃, 3 hours; At last carrier is connected with fragment, reaction system is 10 microlitres, room temperature 5 hours.After transforming, identify, checking order, obtain hemolysin secretion vector pMohly1-sj23LHDGST.
2, the electroporation of recombinant attenuated Salmonellas transforms:
The Salmonellas electricity changes competent preparation: inoculate fresh attenuation salmonella in 200 ml LB substratum, 37 ℃ of shaking tables are cultured to the OD value between 0.4-0.6, centrifugal 6000g * 5 min collect thalline, with aseptic double-distilled water washing thalline one time, centrifugal 6000 g * 5 min are with the washing thalline of 10% glycerine two times, centrifugal 6000 g * 5 min, resuspended with 500 microlitres, 10% glycerine, packing 50 microlitre/pipes, being used for electricity changes.
Adopt electroporation method that the recombiant vaccine dna vector is transformed in the attenuation salmonella VNP20009: under aseptic condition, the recombinant vectors (secretion of III type or hemolysin secretion) that 0.5-5 μ g is built is added in the electricity commentaries on classics competence, behind the mixing, transfer in the electric revolving cup of 0.2 μ M, be used for electric shock, electric commentaries on classics condition is 1.8 KV, 500 Ω, 2 μ F, electricity is coated with ammonia benzyl plate screening after changeing, and the bacterium colony that grows is the reorganization bacterium.
3, immune programme for children and method:
6-8 female mice BALB/C in age in week divides into groups every mouse oral 1 * 10 according to 20 every group
9The unloaded bacterium group of cfu bacterium dose inoculation, reorganization III type secretion group, reorganization hemolysin secretion group, the oral 0.2 ml PBS of natural control group.Oral immunity three times, at interval biweekly, schistosomicide was carried out in one week in the back for the third time.
4, recombiant vaccine bacterium titre in vivo detects:
BALB/C mice oral 10
9Cfu reorganization III type secretion bacterium, reorganization hemolysin secretion bacterium and the wild attenuation salmonella that does not transform, got spleen and mesenteric lymph nodes respectively at 3 days, 10 days, 20 days, weigh, according to the 5:1(PBS volume: the ratio internal organs quality) adds PBS and carries out homogenate, then homogenate is carried out gradient dilution, be applied to ammonia benzyl flat board, titre according to reorganization bacterium in longer colony number and the internal organs Mass Calculation internal organs, the wild attenuation salmonella of non-conversion is coated with does not normally have the LB of resistance flat board, calculates total count with this.
5, sj23LHDGST antibody test:
The preparation of immune serum: BALB/C mice is according to above-mentioned immune programme for children inoculated bacteria vaccine, a week after the last immunity, and tail vein blood, after room temperature left standstill 2-4 hour, centrifugal 5 minutes of 5000 rpm collected serum;
The antigenic bag quilt of sj23LHDGST: utilize 50 mM, the carbonate buffer solution of pH9.6 is according to 10 μ g/ml dilution antigen, and every hole drips 100 μ l, and 4 degree bags are spent the night, and PBST gives a baby a bath on the third day after its birth and is used for detecting after time (5 minutes once).
ELISA measures antibody titers: bag is adopted the BSA of 200 μ l 3% 37 degree sealings 3 hours by the every hole of plate, through PBST give a baby a bath on the third day after its birth time (5 minutes once), use 100 μ l PBS with serum gradient dilution 50 extraordinarily in the hand-hole then, 37 ℃ were reacted 1-2 hour, PBST give a baby a bath on the third day after its birth time (5 minutes once), adding HRP(horseradish peroxidase) link coupled goat anti-mouse igg two is anti-, 37 ℃ were reacted 1 hour, PBST give a baby a bath on the third day after its birth time (5 minutes once), add the tmb substrate colour developing, the 2 M vitriol oils stop, and measure 450 nM absorb light.
6, IFN-γ detects:
Last is after one week of immunity, get three mouse spleens for every group, make single cell suspension under the aseptic condition, RPMI 1640 adjustment cell concns with 10% foetal calf serum behind the erythrocyte splitting are 2 * 106/ml, join in 96 orifice plates according to every hole 100 microlitres, stimulate 72 hours with 10 mcg/ml sj23LHD, 10 mcg/ml GST, 10 mcg/ml Con A respectively, the collecting cell culture supernatant, adopt IFN-γ ELISA test kit, to specifications, measure IFN-γ content
7, immanoprotection action is observed:
In a week after the last immunity, every mouse infects 40 ± 2 schistosoma japonicum cercariaes through skin of abdomen, infects the back and cuts open in 6 weeks extremely, collects adult with perfusion and calculates worm lotus mean; Take by weighing liver 0.5 g, add 10 ml5% KOH, get 0.1 ml smear behind 37 ℃ of digestion 5 h, at the microscopically counting, promptly every gram hepatic tissue worm's ovum number (EPG) is organized the plasmid immune protective efficiency with worm reduction rate and the evaluation of every gram liver egg reduction rate.Calculation formula is as follows: worm reduction rate (%)=[(control group worm lotus mean-experimental group worm lotus mean)/control group worm lotus mean] * 100%; Egg reduction rate (%)=[(control group EPG-experimental group EPG)/control group EPG] * 100%.
1) and hemolysin excretory system (Figure 1A: 2) the attenuation salmonella VNP20009 of embodiment of the invention utilization safety is carrier, adopts two kinds of excretory systems that the two valency antigen sj23LHD-GST of Schistosoma japonicum are carried out secreting, expressing: III type excretory system (Figure 1A:.After the method for utilizing electricity to transform transforms attenuation salmonella, obtain the unloaded bacterium that the III type is secreted bacterium, the hemolysin of recombinating is secreted bacterium and transformed unloaded plasmid of recombinating, adopt western bloting technology for detection the bacterium of respectively recombinating to antigenic secretion situation, the result is presented in the nutrient solution supernatant of unloaded bacterium and does not detect sj23LHD-GST antigen (Figure 1B: 1); Reorganization hemolysin secretion bacteria culture fluid supernatant (Figure 1B: 2) and reorganization III type secrete (Figure 1B: 4) all can detect antigen in the bacterium anaerobism culture supernatant; Because what III type excretory system adopted is the nirB promotor of weary oxygen regulation and control, therefore in reorganization III type secretion bacterium aerobic culture supernatant, do not detect antigen (Figure 1B: 3).Carry situation in order further to prove the antigen of reorganization bacterium in antigen presenting cell-scavenger cell, the present invention adopts the EGFP(green fluorescent protein) as analogue antigen, respectively recombinate bacterium behind Infection in Vitro macrophage system RAW264.7 when what carry EGFP, after infecting unloaded bacterium, do not observe fluorescence (Fig. 1 C:1) in the scavenger cell born of the same parents; After infecting reorganization III type secretion bacterium (Fig. 1 C:2) and reorganization hemolysin secretion bacterium (Fig. 1 C:3), in the scavenger cell born of the same parents, all can detect fluorescence.
In order to estimate the plasmid stability of the bacterium of respectively recombinating, each recombinate bacterium and the unconverted bacterium of wildness are according to 1 * 10
9Cfu/ oral dose is only inoculated the 5-7 Balb/C mouse in age in week.After inoculation, separate peripheral immune organ spleen and lymphoglandula in 3,10,20 days, calculate the titre of bacterium in organ of respectively recombinating.In spleen, with respect to wild bacterium (Fig. 2 A:1), reorganization hemolysin secretion bacterium (Fig. 2 A:2) and reorganization III type secretion bacterium (Fig. 2 A:3) all reduce less than obvious in each time point titre; Also be like this equally in lymphoglandula, with respect to wild bacterium (Fig. 2 B:1), reorganization hemolysin secretion bacterium (Fig. 2 B:2) and reorganization III type secretion bacterium (Fig. 2 B:3) all reduce less than obvious in each time point titre.
The present invention has further estimated the immune response at antigen molecule that produces behind each oral each recombiant vaccine bacterium.Mouse oral immunity PBS, unloaded bacterium and recombiant vaccine bacterium three times, at interval biweekly, a week is got serum after the last immunity, and the antibody of estimating anti-sj23LHD-GST in the serum produces.With respect to unloaded bacterium (Fig. 3 A:1), reorganization III type secretion bacterium (Fig. 3 A:2) and reorganization hemolysin secretion bacterium (Fig. 3 A:3) can both produce at the special antibody of antigen molecule, and the III of wherein recombinating type secretion bacterium (Fig. 3 A:2) antibody titer is the highest.Owing to the oral vaccine that with the Salmonellas is carrier is mainly induced body generation Th1 reaction, so the present invention is also respectively with reorganization sj23LHD, reorganization GST, and positive control medicine Con A immune stimulatory mouse boosting cell, analyze the generation of splenocyte secrete cytokines IFN-γ, compare with unloaded bacterium (Fig. 3 B:2) immunity with PBS immunity (Fig. 3 B:1), reorganization hemolysin secretion bacterium (Fig. 3 B:3) and reorganization III type secretion bacterium (Fig. 3 B:4) immune mouse spleen cell can both produce higher IFN-γ, compare reorganization hemolysin secretion bacterium (Fig. 3 B:3), the IFN-γ that reorganization III type secretion bacterium (Fig. 3 B:4) immune mouse produces is higher.
The present invention has also further estimated the oral protection of bacterium to schistosomicide of respectively recombinating: mouse oral immunity PBS, unloaded bacterium and recombiant vaccine bacterium three times; at interval biweekly; all subcutaneous abdomen infect 40 of schistosoma japonicum cercariaes after the last immunity; infect to calculate in back 42 days and become borer population in the mouse body; worm reduction rate; liver worm's ovum number, egg reduction rate.Immunity PBS mouse become borer population be 30 ± 3 (Fig. 4 A:1) becomes borer population with unloaded bacterium be 26 ± 3 (Fig. 4 A:2), it is 20 ± 3 (Fig. 4 A:3) that reorganization hemolysin secretion bacterium becomes borer population, it is 17 ± 3 (Fig. 4 A:4) that reorganization III type is secreted bacterium one-tenth borer population; With respect to the PBS immunity, the worm reduction rate of unloaded bacterium is 14.98%(Fig. 4 B:1), worm reduction rate 32.93%(Fig. 4 B:2 of reorganization hemolysin secretion bacterium), the worm reduction rate of reorganization III type secretion bacterium is 41.69%(Fig. 4 B:3); Immunity PBS mouse liver worm's ovum number is 122699 ± 34214(Fig. 4 C:1), unloaded bacterium liver worm's ovum number is 100398 ± 21669(Fig. 4 C:2), reorganization hemolysin secretion bacterium liver worm's ovum number is 73058 ± 25481(Fig. 4 C:3), reorganization III type secretion bacterium liver worm's ovum number is 51889 ± 12888(Fig. 4 C:4); With respect to the PBS immunity, the egg reduction rate of unloaded bacterium is 18.17%(Fig. 4 D:1), reorganization hemolysin secretion bacterium egg reduction rate is 40.46%(Fig. 4 D:2), reorganization III type secretion bacterium egg reduction rate is 57.71%(Fig. 4 D:3).
In sum, the invention provides a kind of Schistosoma japonicum oral vaccine and application thereof that utilizes the attenuation salmonella excretory system to express, this blood fluke vaccine oral vaccine is carrier with the attenuation salmonella, and the mode by the secreting, expressing vaccine antigen breaks through the restriction of vesica structure for antigen presentation.The attenuation salmonella oral vaccine submission system of secreting, expressing of the present invention can express and submission antigen in antigen presenting cell effectively, and has body internal stability preferably.This vaccine submission system can activate the immune response of body generation at antigen molecule, obtains excellent protection efficient.
Above-described specific embodiment; purpose of the present invention, technical scheme and beneficial effect are further described; institute is understood that; the above only is specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any modification of being made, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
＜110〉OrganizationName: Nanjing University
＜120〉Title: a kind of Schistosoma japonicum oral vaccine and application of attenuation salmonella secreting, expressing
<160>?14
<210>?1
<211>?Length?:?312
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tcagctgcag?gtagcttcga?tgttaaagag?gaaagaactg?cagcttcttt?attgcagttg 120
tccggtaatg?ccagtgattt?ttcatatgga?cggaactcaa?taaccctgac?cacatcagca 180
taa 183
<210>?4
<211>?Length?:?382
SequenceName?:?4
SequenceDescription: nirB promotor
<212>?Type?:?DNA
<213>?OrganismName?: Escherichia?coli
<400>?PreSequenceString?:
ggttaccggc?ccgatcgttg?aacatagcgg?tccgcaggcg?gcactgctta?cagcaaacgg 60
tctgtacgct?gtcgtctttg?tgatgtgctt?cctgttaggt?ttcgtcagcc?gtcaccgtca 120
gcataacacc?ctgacctctc?attaattgct?catgccggac?ggcactatcg?tcgtccggcc 180
ttttcctctc?ttcccccgct?acgtgcatct?atttctataa?acccgctcat?tttgtctatt 240
ttttgcacaa?acatgaaata?tcagacaatt?ccgtgactta?agaaaattta?tacaaatcag 300
caatataccc?attaaggagt?atataaaggt?gaatttgatt?tacatcaata?agcggggttg 360
ctgaatcgtt?aaggtaggcg?gt 382
<210>?5
<211>?Length?:?891
SequenceName?:?5
SequenceDescription?:?sj23LHD-GST
<212>?Type?:?DNA
<213>?OrganismName?:?Schistosoma japonicum
<400>?PreSequenceString?:
tacaaggata?aaatcgatga?cgaaattaat?acactaatga?ctggtgctct?ggaaaatcca 60
aacgaggaaa?taacggcaac?catggataag?atacaaacat?cattccattg?ttgtggagtc 120
aaaggtccag?acgattataa?agggaatgtg?ccagcatcat?gtaaagaagg?gcaagaagtt 180
tatgttcagg?gttgtctatc?tgtctttagt?gcattcttaa?aacgcaactt?aatgtcccct 240
atactaggtt?attggaaaat?taagggcctt?gtgcaaccca?ctcgacttct?tttggaatat 300
cttgaagaaa?aatatgaaga?atggcatttg?tatgagcgcg?atgaaggtga?taaatggcga 360
aacaaaaagt?ttgaattggg?tttggagttt?cccaatcttc?cttattatat?tgatggtgat 420
gttaaattaa?cacagtctat?ggccatcata?cgttatatag?ctgacaagca?caacatgttg 480
ggtggttgtc?caaaagagcg?tgcagagatt?tcaatgcttg?aaggagcggt?tttggatatt 540
agatacggtg?tttcgagaat?tgcatatagt?aaagactttg?aaactctcaa?agttgatttt 600
cttagcaagc?tacctgaaat?gctgaaaatg?ttcgaagatc?gtttatgtca?taaaacatat 660
ttaaatggtg?atcatgtaac?ccatcctgac?ttcatgttgt?atgacgctct?tgatgttgtt 720
ttatacatgg?acccaatgtg?cctggatgcg?ttcccaaaat?tagtttgttt?taaaaaacgt 780
attgaagcta?tcccacaaat?tgataagtac?ttgaaatcca?gcaagtatat?agcatggcct 840
ttgcagggct?ggcaagccac?gtttggtggt?ggcgaccatc?ctccaaaata?a 891
<210>?6
<211>?Length?:?29
SequenceName?:?6
SequenceDescription: III type secretion signal sopE P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccggatcca?tgactaacat?aacactatc 29
<210>?7
<211>?Length?:?26
SequenceName?:?7
SequenceDescription: III type secretion signal sopE P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
aaaggtaccc?ggatctttac?tcgcat 26
<210>?8
<211>?Length?:?26
SequenceName?:?8
SequenceDescription?:?nirB?P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccctcgagg?gttaccggcc?cgatcg 26
<210>?9
<211>?Length?:?29
SequenceName?:?9
SequenceDescription?:?nirB?P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccggatcca?ccgcctacct?taacgattc 29
<210>?10
<211>?Length?:?31
SequenceName?:?10
SequenceDescription: III type secretion sj23LHD-GST P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
aaaggtacca?tgtacaagga?taaaatcgat?g 31
<210>?11
<211>?Length?:?36
SequenceName?:?11
SequenceDescription: III type secretion sj23LHD-GST P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
taagttgcgt?tttaagaatg?ctagtatagg?ggacat 36
<210>?12
<211>?Length?:?36
SequenceName?:?12
SequenceDescription: III type secretion sj23LHD-GST P3
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
ttcttaaaac?gcaacttaat?gtcccctata?ctaggt 36
<210>?13
<211>?Length?:?29
SequenceName?:?13
SequenceDescription: III type secretion sj23LHD-GST P4
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccaagcttt?tattttggag?gatggtcgc 29
<210>?14
<211>?Length?:?27
SequenceName?:?14
SequenceDescription: I type secretion si23LHD-GST P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
ccaatgcatc?gtacaaggat?aaaatcg 27
<210>?15
<211>?Length?:?27
SequenceName?:?15
SequenceDescription: I type secretion si23LHD-GST P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
ccaatgcatc?ttttggagga?tggtcgc 27
＜110〉OrganizationName: Nanjing University
＜120〉Title: a kind of Schistosoma japonicum oral vaccine and application of attenuation salmonella secreting, expressing
<160>?14
<210>?1
<211>?Length?:?312
SequenceName?:?1
SequenceDescription: III type secretion signal sopE
<212>?Type?:?DNA
<213>?OrganismName?:?Salmonella?typhimurium
<400>?PreSequenceString?:
atgactaaca?taacactatc?cacccagcac?tacagaatcc?atagaagtga?cgttgaacca 60
gtaaaagaaa?aaacaacgga?gaaggacatt?tttgcaaaaa?gtattactgc?cgttagaaat 120
agctttatca?gcctgtcgac?gagtctgtca?gatcgtttta?gcctgcatca?acaaacagac 180
ataccgacta?cccattttca?tcgtgggaac?gcttctgagg?gtagggcggt?attaaccagt 240
aaaactgtta?aagattttat?gctgcaaaag?ctcaatagtc?tggatatcaa?aggtaatgcg 300
agtaaagatc?cg 312
<210>?2
<211>?Length?:?102
SequenceName?:?2
SequenceDescription: I type secretion signal hlyN
<212>?Type?:?DNA
<213>?OrganismName?: Escherichia?coli
<400>?PreSequenceString?:
atgacaacaa?taaccactgc?acaaattaaa?agcacactgc?agtctgcaaa?gcaatccgct 60
gcaaataaat?tgcactcagc?aggacaaagc?acgaaagatg?ca 102
<210>?3
<211>?Length?:?183
SequenceName?:?3
SequenceDescription: I type secretion signal hlyC
<212>?Type?:?DNA
<213>?OrganismName?: Escherichia?coli
<400>?PreSequenceString?:
ttagcctatg?gaagtcaggg?tgatcttaat?ccattaatta?atgaaatcag?caaaatcatt 60
tcagctgcag?gtagcttcga?tgttaaagag?gaaagaactg?cagcttcttt?attgcagttg 120
tccggtaatg?ccagtgattt?ttcatatgga?cggaactcaa?taaccctgac?cacatcagca 180
taa 183
<210>?4
<211>?Length?:?382
SequenceName?:?4
SequenceDescription: nirB promotor
<212>?Type?:?DNA
<213>?OrganismName?: Escherichia?coli
<400>?PreSequenceString?:
ggttaccggc?ccgatcgttg?aacatagcgg?tccgcaggcg?gcactgctta?cagcaaacgg 60
tctgtacgct?gtcgtctttg?tgatgtgctt?cctgttaggt?ttcgtcagcc?gtcaccgtca 120
gcataacacc?ctgacctctc?attaattgct?catgccggac?ggcactatcg?tcgtccggcc 180
ttttcctctc?ttcccccgct?acgtgcatct?atttctataa?acccgctcat?tttgtctatt 240
ttttgcacaa?acatgaaata?tcagacaatt?ccgtgactta?agaaaattta?tacaaatcag 300
caatataccc?attaaggagt?atataaaggt?gaatttgatt?tacatcaata?agcggggttg 360
ctgaatcgtt?aaggtaggcg?gt 382
<210>?5
<211>?Length?:?891
SequenceName?:?5
SequenceDescription?:?sj23LHD-GST
<212>?Type?:?DNA
<213>?OrganismName?:?Schistosoma japonicum
<400>?PreSequenceString?:
tacaaggata?aaatcgatga?cgaaattaat?acactaatga?ctggtgctct?ggaaaatcca 60
aacgaggaaa?taacggcaac?catggataag?atacaaacat?cattccattg?ttgtggagtc 120
aaaggtccag?acgattataa?agggaatgtg?ccagcatcat?gtaaagaagg?gcaagaagtt 180
tatgttcagg?gttgtctatc?tgtctttagt?gcattcttaa?aacgcaactt?aatgtcccct 240
atactaggtt?attggaaaat?taagggcctt?gtgcaaccca?ctcgacttct?tttggaatat 300
cttgaagaaa?aatatgaaga?atggcatttg?tatgagcgcg?atgaaggtga?taaatggcga 360
aacaaaaagt?ttgaattggg?tttggagttt?cccaatcttc?cttattatat?tgatggtgat 420
gttaaattaa?cacagtctat?ggccatcata?cgttatatag?ctgacaagca?caacatgttg 480
ggtggttgtc?caaaagagcg?tgcagagatt?tcaatgcttg?aaggagcggt?tttggatatt 540
agatacggtg?tttcgagaat?tgcatatagt?aaagactttg?aaactctcaa?agttgatttt 600
cttagcaagc?tacctgaaat?gctgaaaatg?ttcgaagatc?gtttatgtca?taaaacatat 660
ttaaatggtg?atcatgtaac?ccatcctgac?ttcatgttgt?atgacgctct?tgatgttgtt 720
ttatacatgg?acccaatgtg?cctggatgcg?ttcccaaaat?tagtttgttt?taaaaaacgt 780
attgaagcta?tcccacaaat?tgataagtac?ttgaaatcca?gcaagtatat?agcatggcct 840
ttgcagggct?ggcaagccac?gtttggtggt?ggcgaccatc?ctccaaaata?a 891
<210>?6
<211>?Length?:?29
SequenceName?:?6
SequenceDescription: III type secretion signal sopE P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccggatcca?tgactaacat?aacactatc 29
<210>?7
<211>?Length?:?26
SequenceName?:?7
SequenceDescription: III type secretion signal sopE P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
aaaggtaccc?ggatctttac?tcgcat 26
<210>?8
<211>?Length?:?26
SequenceName?:?8
SequenceDescription?:?nirB?P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccctcgagg?gttaccggcc?cgatcg 26
<210>?9
<211>?Length?:?29
SequenceName?:?9
SequenceDescription?:?nirB?P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccggatcca?ccgcctacct?taacgattc 29
<210>?10
<211>?Length?:?31
SequenceName?:?10
SequenceDescription: III type secretion sj23LHD-GST P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
aaaggtacca?tgtacaagga?taaaatcgat?g 31
<210>?11
<211>?Length?:?36
SequenceName?:?11
SequenceDescription: III type secretion sj23LHD-GST P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
taagttgcgt?tttaagaatg?ctagtatagg?ggacat 36
<210>?12
<211>?Length?:?36
SequenceName?:?12
SequenceDescription: III type secretion sj23LHD-GST P3
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
ttcttaaaac?gcaacttaat?gtcccctata?ctaggt 36
<210>?13
<211>?Length?:?29
SequenceName?:?13
SequenceDescription: III type secretion sj23LHD-GST P4
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
cccaagcttt?tattttggag?gatggtcgc 29
<210>?14
<211>?Length?:?27
SequenceName?:?14
SequenceDescription: I type secretion si23LHD-GST P1
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
ccaatgcatc?gtacaaggat?aaaatcg 27
<210>?15
<211>?Length?:?27
SequenceName?:?15
SequenceDescription: I type secretion si23LHD-GST P2
<212>?Type?:?DNA
<213>?OrganismName?:?Artificial
<400>?PreSequenceString?:
ccaatgcatc?ttttggagga?tggtcgc 27
Claims (9)
1. blood fluke vaccine Salmonellas secretion expression carrier, it is characterized in that by the control of bacterium promotor, Salmonellas secretion effect protein and secretion signal recombinant salmonella expression vector guiding, the secreting, expressing schistosome antigen thereof.
2. a kind of blood fluke vaccine Salmonellas secretion expression carrier according to claim 1 is characterized in that the bacterium promotor is the nitrite reductase promotor or the Salmonellas hemolysin promotor in intestinal bacteria source.
3. a kind of blood fluke vaccine Salmonellas secretion expression carrier according to claim 1 is characterized in that lacking secretion inducing expression blood fluke vaccine under the oxygen microenvironment in antigen presenting cell.
4. a kind of blood fluke vaccine Salmonellas secretion expression carrier according to claim 1 is characterized in that stablizing in vivo, exists constantly, and expression vector is not lost.
5. a kind of blood fluke vaccine Salmonellas secretion expression carrier according to claim 1, it is characterized in that containing Salmonellas secretion effect protein and secretion signal thereof, described Salmonellas secretion effect protein and secretion signal thereof are Salmonellas III type secretion effect protein and secretory signal sequence thereof, especially Salmonellas outer membrane protein E N end 1-104 aminoacid sequence.
6. a kind of blood fluke vaccine Salmonellas secretion expression carrier according to claim 1, it is characterized in that containing Salmonellas secretion effect protein and secretion signal thereof, described Salmonellas secretion effect protein and secretion signal thereof are hemolysin secretion effect protein and secretory signal sequence thereof, and described hemolysin secretion effect protein is hlyA, hlyB, hlyC, hlyD; Described secretory signal sequence is 34 amino acid of Salmonellas hlyA albumen n end and 61 aminoacid sequences of C end.
7. the application method of a kind of blood fluke vaccine Salmonellas secretion expression carrier according to claim 1 is characterized in that and will adopt comparatively safe attenuated bacteria strain as delivery vector that described attenuated bacteria strain is attenuation salmonella VNP20009.
8. the application method of a kind of blood fluke vaccine Salmonellas secretion expression carrier according to claim 1 is characterized in that adopting oral methods to carry out the antigen delivery of recombiant vaccine submission bacterium.
9. the application of the described a kind of blood fluke vaccine Salmonellas secretion expression carrier of claim 1 in preparation schistosomicide oral vaccine.
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| CN201110232776.2A CN102277373B (en) | 2011-08-15 | 2011-08-15 | Schistosoma japonica vaccine expression vector for attenuated salmonella secretion expression and use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103204936A (en) * | 2013-01-25 | 2013-07-17 | 天津商业大学 | Preparation method for polyclonal antibody to salmonella effect protein SopB |
| CN104805113A (en) * | 2015-05-06 | 2015-07-29 | 南昌大学 | Application of attenuated salmonella typhimurium VNP20009 in gene expression |
| WO2022087854A1 (en) * | 2020-10-26 | 2022-05-05 | 南京吉芮康生物科技研究院有限公司 | Sars-cov-2 vaccine antigen presenting system of ntd domain protein secreted and expressed by attenuated salmonella typhimurium, and use thereof |
| CN114703214A (en) * | 2022-03-03 | 2022-07-05 | 南京吉芮康生物科技研究院有限公司 | Novel loss-resistant spatiotemporal controllable expression plasmid and application thereof |
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| CN102335421A (en) * | 2011-08-03 | 2012-02-01 | 南京大学 | Attenuated salmonella inducible secretory expression oral vaccine presentation system and application thereof |
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| CN102335421A (en) * | 2011-08-03 | 2012-02-01 | 南京大学 | Attenuated salmonella inducible secretory expression oral vaccine presentation system and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103204936A (en) * | 2013-01-25 | 2013-07-17 | 天津商业大学 | Preparation method for polyclonal antibody to salmonella effect protein SopB |
| CN103204936B (en) * | 2013-01-25 | 2014-06-18 | 天津商业大学 | Preparation method for polyclonal antibody to salmonella effect protein SopB |
| CN104805113A (en) * | 2015-05-06 | 2015-07-29 | 南昌大学 | Application of attenuated salmonella typhimurium VNP20009 in gene expression |
| WO2022087854A1 (en) * | 2020-10-26 | 2022-05-05 | 南京吉芮康生物科技研究院有限公司 | Sars-cov-2 vaccine antigen presenting system of ntd domain protein secreted and expressed by attenuated salmonella typhimurium, and use thereof |
| CN114480462A (en) * | 2020-10-26 | 2022-05-13 | 南京吉芮康生物科技研究院有限公司 | Novel coronavirus vaccine antigen presentation system for secretion expression of NTD structural domain protein by attenuated salmonella and application thereof |
| CN114703214A (en) * | 2022-03-03 | 2022-07-05 | 南京吉芮康生物科技研究院有限公司 | Novel loss-resistant spatiotemporal controllable expression plasmid and application thereof |
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| CN102277373B (en) | 2014-07-02 |
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